Ghrelin對(duì)AngⅡ 誘導(dǎo)的心肌細(xì)胞凋亡的影響及機(jī)制

楊春艷,馮朝暉,楊 萍,任 平

(1.吉林大學(xué)中日聯(lián)誼醫(yī)院 心內(nèi)科,吉林 長(zhǎng)春130033;2.吉林大學(xué)第一醫(yī)院 胸外科)

Ghrelin對(duì)AngⅡ 誘導(dǎo)的心肌細(xì)胞凋亡的影響及機(jī)制

楊春艷1,馮朝暉1,楊 萍1,任 平2*

(1.吉林大學(xué)中日聯(lián)誼醫(yī)院 心內(nèi)科,吉林 長(zhǎng)春130033;2.吉林大學(xué)第一醫(yī)院 胸外科)

目的闡明Ghrelin對(duì)血管緊張素Ⅱ( AngⅡ)誘導(dǎo)的心肌細(xì)胞凋亡的影響及其可能機(jī)制。方法體外培養(yǎng)H9C2心肌細(xì)胞，分為空白對(duì)照組、Ang Ⅱ組、Ang Ⅱ+Ghrelin組及單純Ghrelin組，采用MTT法檢測(cè)細(xì)胞存活率，TUNEL染色觀察心肌細(xì)胞凋亡情況，并應(yīng)用RT-PCR法檢測(cè)Bcl-2、Bax、Caspase-3、1型及2型Ang Ⅱ 受體(AT1R，AT2R)mRNA表達(dá)。結(jié)果與空白對(duì)照組相比，Ang Ⅱ組心肌細(xì)胞存活率明顯下降；細(xì)胞凋亡數(shù)目明顯增加；Caspase-3及促凋亡分子Bax表達(dá)明顯增加，而抗凋亡分子Bcl-2表達(dá)明顯減少；AT1R 及AT2R表達(dá)均明顯增加，AT1R增加尤為顯著。與Ang Ⅱ組相比，Ghrelin+Ang Ⅱ組心肌細(xì)胞存活率明顯增加；細(xì)胞凋亡數(shù)目明顯減少；Caspase-3及促凋亡分子Bax表達(dá)明顯減少，而抗凋亡分子Bcl-2表達(dá)明顯增加；AT1R表達(dá)明顯減少，而AT2R表達(dá)無(wú)明顯變化。結(jié)論AT1R及AT2R均參與Ang Ⅱ誘導(dǎo)心肌細(xì)胞凋亡進(jìn)程，Ghrelin可抑制Ang Ⅱ誘導(dǎo)的心肌細(xì)胞凋亡，其機(jī)制可能與Ghrelin下調(diào)通過(guò)下調(diào)AT1R表達(dá)有關(guān)。

Ghrelin;血管緊張素Ⅱ;心肌細(xì)胞凋亡;血管緊張素Ⅱ受體

(ChinJLabDiagn,2017,21:1982)

心肌細(xì)胞凋亡可導(dǎo)致心肌細(xì)胞數(shù)目的減少進(jìn)而影響心肌收縮性，并可引起心肌細(xì)胞代償性肥大及纖維化，促進(jìn)心血管系統(tǒng)疾病的進(jìn)展。因此，抑制或改善心肌細(xì)胞凋亡是是臨床關(guān)心的一個(gè)重要課題，探尋可抑制心肌細(xì)胞凋亡的有效藥物，并明確其抑制心肌細(xì)胞的凋亡分子機(jī)制具有重要意義。促生長(zhǎng)激素釋放多肽(Ghrelin)是生長(zhǎng)激素促分泌素(GHS)受體的內(nèi)源性配體，具有多種生物學(xué)活性，包括刺激生長(zhǎng)激素(GH)分泌、調(diào)節(jié)代謝、促進(jìn)攝食與肥胖等[1]。目前，有研究發(fā)現(xiàn)Ghrelin及其受體可在心臟表達(dá)[2]，并與心臟上的結(jié)合位點(diǎn)具有高度親合力[3-5]，提示心臟為Ghrelin作用的靶器官。Ghrelin還具有增加心肌收縮力、舒張血管、保護(hù)內(nèi)皮細(xì)胞、改善心肌能量代謝以及預(yù)防保護(hù)心梗后心衰的形成等多種心血管保護(hù)作用[6-9]。然而，Ghrelin發(fā)揮心血管保護(hù)作用的細(xì)胞及分子機(jī)制尚有待進(jìn)一步研究。H9c2心肌細(xì)胞是從胚胎大鼠心室肌中分離克隆的一個(gè)細(xì)胞系[10]，以往的研究表明，它是體外研究心肌細(xì)胞凋亡模型的理想細(xì)胞[11,12]。在本研究中，我們培養(yǎng)H9c2細(xì)胞，探討Ghrelin對(duì)血管緊張素Ⅱ(Ang Ⅱ)誘導(dǎo)的H9c2細(xì)胞凋亡的影響及其可能機(jī)制，為臨床應(yīng)用Ghrelin治療心血管疾病提供實(shí)驗(yàn)依據(jù)。

1 材料與方法

1.1主要試劑?；疓hrelin購(gòu)自于中肽生物科技有限公司，Ang Ⅱ 與MTT購(gòu)自于美國(guó)Sigma-Aldrich公司 (St.Louis,MO,USA)，TUNEL檢測(cè)試劑盒購(gòu)自羅氏公司(South San Francisco,California,USA)。

1.2H9c2細(xì)胞培養(yǎng)及分組H9c2 細(xì)胞用含10%血清的高糖DMEM培養(yǎng)液培養(yǎng),于培養(yǎng)瓶，37℃，5%CO2培養(yǎng)箱孵育，傳至2-3代后用于實(shí)驗(yàn)。實(shí)驗(yàn)分為以下四組：空白對(duì)照(Con)組(培養(yǎng)液對(duì)照)，Ang Ⅱ組(培養(yǎng)液+ 10-7mol/L Ang Ⅱ )，Ang Ⅱ+ghrelin組(培養(yǎng)液+10-7mol/L Ang Ⅱ +10-7 mol/L Ghrelin )，Ghrelin組(培養(yǎng)液+ 10-7mol/L Ghrelin)。

1.3MTT法檢測(cè)H9c2心肌細(xì)胞活性將濃度為5-6×105/ml的H9c2心肌細(xì)胞懸液接種于96孔板，待細(xì)胞長(zhǎng)至70%-80%時(shí)給予Ang Ⅱ及Ghrelin干預(yù)，并于加藥刺激24 h后每孔加入20 μl MTT (5 mg/ml)，于37℃細(xì)胞培養(yǎng)箱繼續(xù)孵育4 h。去除細(xì)胞上清，每孔加入150 μl 二甲基亞砜(DMSO)，避光搖晃10 min，于490 nm 處測(cè)細(xì)胞吸光度，并計(jì)算細(xì)胞存活百分率。

1.4原位末端標(biāo)記檢測(cè)法(TUNEL)染色檢測(cè)H9c2心肌細(xì)胞凋亡將生長(zhǎng)于玻片上的四組心肌細(xì)胞應(yīng)用TUNEL試劑進(jìn)行染色，具體步驟按TUNEL說(shuō)明書(shū)進(jìn)行。染色結(jié)束后于暗室光鏡下觀察，細(xì)胞核呈綠色熒光即為T(mén)UNEL陽(yáng)性細(xì)胞。

1.5RT-PCR檢測(cè)Bcl-2、Bax、Caspase-3、AT1R及AT2R的mRNA表達(dá)提取總RNA及逆轉(zhuǎn)錄反應(yīng)：Trizols提取各組H9c2細(xì)胞的總RNA，分光光度計(jì)測(cè)定RNA的濃度和純度，，然后取2 μg RNA進(jìn)行逆轉(zhuǎn)錄,合成cDNA后擴(kuò)增30個(gè)循環(huán)。所有引物均由上海生物工程技術(shù)有限公司合成,序列見(jiàn)表 1。

表1 RT-PCR引物合成表

2 結(jié)果

2.1Ghrelin及AngⅡ?qū)9c2心肌細(xì)胞凋亡的影響

應(yīng)用Ang Ⅱ及Ghrelin干預(yù)H9c2心肌細(xì)胞24小時(shí)后，MTT法測(cè)心肌細(xì)胞存活率發(fā)現(xiàn)：與Con組相比，給予Ang Ⅱ刺激后H9c2心肌細(xì)胞存活率明顯下降(80.5±1.6%)，有顯著性差異(Plt;0.01)； Ghrelin干預(yù)組細(xì)胞的存活率無(wú)明顯改變(99.7±4.1%)，差異無(wú)統(tǒng)計(jì)學(xué)意義(Pgt;0.05)；與Ang Ⅱ組相比，Ghrelin 與Ang Ⅱ共同干預(yù)組細(xì)胞存活率明顯增高(90.4±4.7%)，有顯著性差異(Plt;0.01)，見(jiàn)圖1。

進(jìn)一步應(yīng)用TUNEL染色檢測(cè)H9c2心肌細(xì)胞凋亡情況，結(jié)果顯示：與Con組相比，Ang Ⅱ干預(yù)后H9c2心肌細(xì)胞凋亡率明顯增高，有顯著性差異(Plt;0.01)；與Ang Ⅱ組相比，Ghrelin與Ang Ⅱ共同干預(yù)組H9c2心肌細(xì)胞凋亡率明顯降低，有顯著性差異(Plt;0.01)，見(jiàn)圖2及圖3。

**Plt; 0.01 vs.the control group;##Plt; 0.01 vs.the Ang Ⅱ group.

圖1MTT法檢測(cè)心肌細(xì)胞存活率

2.2Ghrelin及AngⅡ?qū)aspase-3表達(dá)的影響

本研究利用Ang Ⅱ誘導(dǎo)H9c2細(xì)胞凋亡，在加藥刺激后24 h，采用RT-PCR檢測(cè)Caspase-3表達(dá)情況。結(jié)果發(fā)現(xiàn)Ang Ⅱ 組較空白對(duì)照組Caspase-3 mRNA表達(dá)水平明顯增加 (Plt;0.01)，而Ghrelin可以明顯下調(diào)Ang Ⅱ誘導(dǎo)的Caspase-3表達(dá) (Plt;0.01)，見(jiàn)圖4。Caspase-3 為與細(xì)胞凋亡呈正相關(guān)的凋亡途徑關(guān)鍵分子，上述結(jié)果提示Ang Ⅱ可通過(guò)促進(jìn)凋亡關(guān)鍵分子Caspase-3表達(dá)而誘導(dǎo)心肌細(xì)胞凋亡，Ghrelin則可通過(guò)下調(diào)Caspase-3表達(dá)而抑制其作用。

圖2 TUNEL染色法檢測(cè)心肌細(xì)胞凋亡(×400)

**Plt;0.01 vs.the control group;##Plt; 0.01 vs.the Ang Ⅱ group.

圖3TUNEL染色法檢測(cè)心肌細(xì)胞凋亡百分率

圖4 RT-PCR檢測(cè)Caspase-3 mRNA表達(dá)

2.3Ghrelin及AngⅡ?qū)cl-2及Bax表達(dá)的影響

Bcl-2及Bax表達(dá)失衡與細(xì)胞凋亡密切相關(guān)。因此，本研究利用Ang Ⅱ誘導(dǎo)H9c2細(xì)胞凋亡，在加藥刺激后24 h，采用RT-PCR檢測(cè)Caspase-3表達(dá)情況。結(jié)果發(fā)現(xiàn)Ang Ⅱ 組較空白對(duì)照組Bax mRNA表達(dá)明顯增加 (Plt;0.01)，Bcl-2 mRNA表達(dá)明顯減少(Plt; 0.01)；而Ghrelin可以明顯下調(diào)Ang Ⅱ誘導(dǎo)的Bax表達(dá)增加(Plt;0.01)，并上調(diào)Ang Ⅱ誘導(dǎo)的Bcl-2表達(dá)減少(Plt; 0.01)，見(jiàn)圖5。上述結(jié)果提示：Ang Ⅱ可能通過(guò)誘導(dǎo)Bcl-2/Bax表達(dá)失衡而導(dǎo)致心肌細(xì)胞凋亡，Ghrelin則可抑制Ang Ⅱ誘導(dǎo)的Bcl-2/Bax表達(dá)失衡發(fā)揮心血管保護(hù)作用。

圖5 RT-PCR檢測(cè)Bcl-2及Bax mRNA表達(dá)

2.4Ghrelin及AngⅡ?qū)T1R及AT2R表達(dá)的影響

為明確AT1R及AT2R在Ang Ⅱ誘導(dǎo)H9c2細(xì)胞凋亡中的作用，實(shí)驗(yàn)采用RT-PCR檢測(cè)AT1R及AT2R mRNA表達(dá)水平，研究結(jié)果顯示，Ang Ⅱ刺激培養(yǎng)的H9c2細(xì)胞24 h后，AT1R及AT2R mRNA較空白對(duì)照組表達(dá)均增加(BothPlt;0.01)，Ghrelin可明顯降低Ang Ⅱ誘導(dǎo)的AT1R表達(dá)增加 (Plt; 0.01)，而對(duì)AT2R表達(dá)無(wú)明顯影響 (Pgt;0.05)，見(jiàn)圖6。

圖6 RT-PCR檢測(cè)AT1R及AT2R mRNA表達(dá)

3 討論

以往的研究表明Ghrelin除刺激生長(zhǎng)激素分泌、調(diào)節(jié)攝食及促進(jìn)新陳代謝外，尚具有許多心血管保護(hù)作用[6-9]。然而，Ghrelin保護(hù)心血管作用的相關(guān)分子機(jī)制尚未完全明確。腎素-血管緊張素-醛固酮系統(tǒng)(RAAS)是維持心血管內(nèi)環(huán)境穩(wěn)定的重要機(jī)制之一，Ang Ⅱ是RAAS的核心分子，在許多心血管疾病進(jìn)程中發(fā)揮重要作用[13-15]。以往的研究表明，Ghrelin可以保護(hù)Ang Ⅱ誘導(dǎo)的內(nèi)皮細(xì)胞損傷[16],Ghrelin可以抑制Ang Ⅱ誘導(dǎo)的人主動(dòng)脈內(nèi)皮細(xì)胞移行[17]。因此，我們推測(cè)：在心血管系統(tǒng)，Ghrelin可能通過(guò)抑制Ang Ⅱ誘導(dǎo)的心肌損傷而發(fā)揮心血管保護(hù)作用。越來(lái)越多的研究表明，Ang Ⅱ可誘導(dǎo)心肌細(xì)胞凋亡[18]，為此，我們用Ang Ⅱ誘導(dǎo)H9c2細(xì)胞凋亡，并用Ghrelin加以干預(yù)，探討Ghrelin對(duì)Ang Ⅱ誘導(dǎo)的H9c2細(xì)胞凋亡的影響。結(jié)果發(fā)現(xiàn)Ang Ⅱ可誘導(dǎo)H9c2 細(xì)胞凋亡，而Ghrelin可抑制Ang Ⅱ誘導(dǎo)的H9c2細(xì)胞凋亡。

Caspase-3 為凋亡途徑的關(guān)鍵分子，其表達(dá)與細(xì)胞凋亡正相關(guān)[19,20]。為進(jìn)一步明確Ghrelin對(duì)心肌細(xì)胞凋亡的影響，本研究進(jìn)一步采用RT-PCR法檢測(cè)Caspase-3表達(dá)情況，結(jié)果發(fā)現(xiàn)Ang Ⅱ 組Caspase-3 mRNA表達(dá)水平明顯增加，而ghrelin可以明顯下調(diào)Ang Ⅱ誘導(dǎo)的Caspase-3表達(dá)。進(jìn)一步證實(shí)Ang Ⅱ可通過(guò)誘導(dǎo)凋亡途徑的關(guān)鍵分子Caspase-3活化而導(dǎo)致心肌細(xì)胞凋亡，Ghrelin則可抑制其作用。

Bcl-2及Bax表達(dá)失衡與細(xì)胞凋亡密切相關(guān)[21-22]，當(dāng)Bcl-2表達(dá)相對(duì)較高而bax表達(dá)較低時(shí)，細(xì)胞活力增強(qiáng)；反之，則可促進(jìn)細(xì)胞凋亡。為探討Ghrlein抑制Ang Ⅱ誘導(dǎo)的H9c2細(xì)胞凋亡的分子機(jī)制，我們還應(yīng)用PCR法檢測(cè)了Bcl-2 與Bax的mRNA表達(dá)水平，結(jié)果發(fā)現(xiàn)在Ang Ⅱ誘導(dǎo)的H9c2細(xì)胞凋亡過(guò)程中Bax的表達(dá)增加而B(niǎo)cl-2表達(dá)減少，導(dǎo)致Bcl-2及Bax表達(dá)失衡，Bcl-2/Bax比值降低，而ghrelin則對(duì)其具有抑制作用。上述結(jié)果提示，ghrelin可能通過(guò)減輕Ang Ⅱ誘導(dǎo)的Bax/Bcl-2比值增加而糾正其表達(dá)失衡，從而抑制Ang Ⅱ誘導(dǎo)的H9c2細(xì)胞凋亡。

Ang Ⅱ是影響心血管系統(tǒng)的重要內(nèi)分泌因子之一，主要通過(guò)1型及2型受體(AT1R，AT2R)發(fā)揮生物學(xué)作用[23]，其中Ang Ⅱ與AT1R的結(jié)合率約50%-70%[24]。Ang Ⅱ通過(guò)AT1R及AT2R發(fā)揮生物學(xué)作用，然而Ang Ⅱ是經(jīng)由AT1R還是AT2R介導(dǎo)心肌細(xì)胞凋亡，仍存在很大爭(zhēng)議[25-27]。為明確AT1R及AT2R在Ang Ⅱ誘導(dǎo)H9c2心肌細(xì)胞凋亡中的作用，研究應(yīng)用RT-PCR法檢測(cè)AT1R及AT2R表達(dá)，結(jié)果發(fā)現(xiàn)Ang Ⅱ刺激后，AT1R及AT2R表達(dá)均上調(diào)，而Ghrelin可下調(diào)AT1R表達(dá)，但對(duì)AT2R表達(dá)無(wú)明顯影響。上述結(jié)果提示：AT1R及AT2R均參與Ang Ⅱ誘導(dǎo)心肌細(xì)胞凋亡過(guò)程，通過(guò)Ghrelin下調(diào)AT1R表達(dá)而抑制Ang Ⅱ誘導(dǎo)的心肌細(xì)胞凋亡，進(jìn)一步提示Ang Ⅱ通過(guò)AT1R介導(dǎo)促凋亡分子Bax及Caspase-3表達(dá)，抑制抗凋亡分子Bcl-2表達(dá)，進(jìn)而誘導(dǎo)心肌細(xì)胞凋亡。此外，Ang Ⅱ干預(yù)后AT2R表達(dá)亦增加，可明確Ghrelin不通過(guò)AT2R抑制Ang Ⅱ誘導(dǎo)的心肌細(xì)胞凋亡，但AT2R是拮抗AT1R的作用還是與AT1R共同介導(dǎo)Ang Ⅱ誘導(dǎo)的心肌細(xì)胞凋亡尚不能明確，仍有待于進(jìn)一步研究。結(jié)合2008年Yanfei等[28]研究發(fā)現(xiàn)的新生乳鼠AT2R過(guò)表達(dá)可誘導(dǎo)心肌細(xì)胞凋亡，推測(cè)本研究發(fā)現(xiàn)的AT2R上調(diào)可能也對(duì)Ang Ⅱ誘導(dǎo)的心肌細(xì)胞凋亡起促進(jìn)作用。

綜上所述，我們目前的體外研究不僅明確了ghrelin 可抑制Ang Ⅱ誘導(dǎo)的H9c2細(xì)胞凋亡，更重要的是，明確了ghrelin 抑制Ang Ⅱ誘導(dǎo)的H9c2細(xì)胞凋亡的可能機(jī)制，即：Ghrelin可通過(guò)下調(diào)H9c2細(xì)胞上的AT1受體，影響凋亡相關(guān)基因bax、Bcl-2及凋亡關(guān)鍵分子caspase - 3的表達(dá)，進(jìn)而抑制Ang Ⅱ誘導(dǎo)的H9c2細(xì)胞凋亡。

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EffectandMechanismofGhrelinonH9c2CardiomyocytesapoptosisinducedbyAngⅡ

YANGChun-yan,FENGZhao-hui,YANGPing,etal.

(Departmentofcardiology,China-JapanUnionHospital,JilinUniversity,Changchun130033,China)

ObjectiveTo clarify the effect and possible mechanism of ghrelin on H9c2 cardiomyocytes apoptosis induced by Angiotensin Ⅱ (Ang Ⅱ)．MethodsH9c2 cells were cultured and divided into blank control group,group of Ang Ⅱ and Ang Ⅱ + Ghrelin and Ghrelin group.MTT was used to detect cell survival,TUNEL staining was used to observe the apoptosis of H9c2 cells,and the RT-PCR was used to detect the mRNA expression of bcl-2,Bax,Caspase- 3,type 1 and type 2 Ang Ⅱ receptors (AT1R,AT2R).ResultsCompared with the control group,the survival rate of H9c2 cells decreased significantly in Ang Ⅱ group.The number of apoptosis was significantly increased.The expression of Caspase-3 and Bax was significantly increased,while the expression of bcl-2 was significantly decreased.AT1R and AT2R were significantly increased,especially AT1R.However,ghrelin could significantly increase the survival rate of myocardial cells,reduce the number of apoptosis,down-regulate the expression of Caspase-3,Bax and AT1R,and up-regulate the expression of bcl-2.Interestingly,there was no significant change in AT2R expression.ConclusionAT1R and AT2R were both involved in the induction of myocardial apoptosis in Ang Ⅱ.Ghrelin inhibited the apoptosis of myocardial cells induced by Ang Ⅱ,and its mechanism could be related to the down-regulation of AT1R.

heart failure;ghrelin;angiotensin Ⅱ;myocardial apoptosis

國(guó)家自然科學(xué)基金(81570360,81400298)

*通訊作者

1007-4287(2017)11-1982-06

R541.6+1

A

楊春艷(1980- )，女，博士，主治醫(yī)師，主要從事心力衰竭發(fā)病機(jī)制及治療研究。

2017-02-13)